Mitochondrial aconitase is highly sensitive to oxidative damage during aging. It reversibly converts citrate to isocitrate in the tricarboxylic acid (TCA) cycle. The cubane [4Fe-4S]2+ cluster of aconitase is essential for its catalytic activity, but it also renders the enzyme highly vulnerable to oxidative stress. We have discovered that isolated wild-type Saccharomyces cerevisiae mitochondria contain a nucleotide (GTP, NAD(P)H and ATP)- dependent machinery for iron-sulfur cluster (ISC) biogenesis of aconitase (Aco1p). The cluster biogenesis occurs in the mitochondrial matrix by a multi-step process requiring multiple components. In Aim I, we will biochemically dissect these steps and determine the nucleotide requirements of each. In Aims II-IV, we will study proteins that we found are directly relevant to nucleotide-dependent processes in ISC biogenesis of Aco1p. These include a GTPase (Mtg1p), a NADH kinase (Pos5p), a NADPH-requiring reductase (Arh1p), and an ATPase (Ssq1p). Mitochondria lacking any of these proteins are deficient in ISC biogenesis of Aco1p and display greatly reduced aconitase activity. However, the causal defects in these mutant mitochondria must be different since these proteins have different functions. Mtg1p is a GTPase in the mitochondrial matrix, and we will test our hypothesis that Mtg1p mediates the effects of matrix GTP on ISC biogenesis of Aco1p (Aim II). Pos5p is a NADH kinase and is required for NADPH production in the matrix. We will determine if the role of Pos5p in ISC biogenesis of Aco1p is mediated by its effects on NADPH levels in the matrix (Aim III). NADPH is likely utilized by reductase(s) such as ferredoxin reductase (Arh1p), which may provide reducing equivalents for one or more steps in cluster biogenesis, and this will be tested as part of Aim III. Ssq1p is an Hsp70 chaperone with ATPase activity in the matrix. Therefore some of the effects of ATP on ISC biogenesis of Aco1p are likely mediated by Ssq1p, and this will be explored (Aim IV). In addition to Ssq1p, yeast mitochondria contain an abundant Hsp70 (Ssc1p) that is involved in protein import. In contrast, human mitochondria contain a single Hsp70 (hSSC1), and thus it may participate in both protein import and ISC biogenesis, and we will test this possibility as part of Aim IV. Aco1p is essential for mitochondrial DNA (mtDNA) stability, and this activity is independent of its enzymatic activity in the TCA cycle. Aim V is to investigate interaction of yeast and human mitochondrial aconitases with mtDNA. We will determine if human mitochondrial aconitase, like yeast Aco1p, is also bifunctional and participates in the maintenance of mtDNA. Nucleotides and/or redox status of the matrix may play a critical role in distributing aconitase between the TCA cycle and mt-nucleoids, and we will examine these possibilities. The mechanism underlying the biogenesis of ISCs of mitochondrial aconitase in yeast is likely to be very similar to that in human. Homologs of most, if not all, yeast proteins that participate in the process also exist in human. Thus, conclusions from yeast studies proposed here will be informative to human physiology. Public Health Relevance: Mitochondrial aconitase requires a 4Fe-4S cluster for its enzymatic activity in the tricarboxylic acid (TCA) cycle and is highly sensitive to oxidative damage during aging. In yeast, aconitase is also essential for the maintenance of mitochondrial DNA, and this activity is independent of its catalytic activity in the TCA cycle. This proposal seeks to investigate the molecular mechanism of Fe-S cluster biogenesis of aconitase and its interaction with mitochondrial DNA.